ABSTRACT The neurotransmitter acetylcholine is widely distributed in the central nervous system and brain, and is a key signaling molecule involved in neural function. Current measurements of this important signaling molecule rely on implanted electrodes or sampling techniques, which often trade temporal or spatial resolution for chemical specificity, or vice versa. Our long-term goal is to apply our MRI-based nanosensor to monitor the distribution and activity of acetylcholine in the brain, and generate a data set that enables comparison of the neural activity differences between healthy and diseased tissue. The objective of this proposal, which is the first step in achieving this goal, is to develop our Acetylcholine Magnetic Resonance Nanosensor (ACh- MRNS) that can continuously and reversibly respond to physiological release of acetylcholine in the brain of live animal models, and pair it with a novel technique for fast, quantitative imaging, namely Dual Contrast - Magnetic Resonance Fingerprinting (DC-MRF). In addition, we will investigate a focused ultrasound technique for enabling nanosensor penetration across the blood brain barrier, to enable detection in the brain after systemic injection of the probe. The mechanism of our sensor is based on a unique pairing of a pH-sensitive MR probe with enzymatic recognition of acetylcholine in a nano-scale scaffold that will detect the local pH changes that are created by the hydrolysis of acetylcholine. The rationale of this research is that when cholinesterase and pH-sensitive MR contrast agents are incorporated into one nanoparticle, the hydrolysis that is catalyzed by cholinesterase reduces the local pH, leading to a change of the T1 and T2 relaxation times of MR contrast agent. We will then combine this highly sensitive and specific MRI agent with our DC-MRF methodology to dynamically and simultaneously track the ACh-MRNS and control agents enabling an in vivo assessment of acetylcholine levels in rodent brains. In this proposal, our multi-disciplinary team will combine our highly sensitive MRI nanosensors with the novel MRF methodology to provide a non- invasive, high resolution, radiation-free imaging platform to study neurotransmitters in vivo. Aims 1 and 2 will focus on the development of the ACh-MRNS sensor and the DC-MRF methodology, respectively. Aim 3 will combine all of these technologies into a comprehensive neural imaging package to provide in vivo MRI assessments of acetylcholine in rodents.